Erythritol is a sugar alcohol that can be found in lichens, hemp leaves, and mushrooms. It is also savored in fermented foods such as wine, soya sauce, or saki (Sasaki, T. (1989) Production technology of erythritol. Nippon Nogeikagaku Kaishi 63: 1130-1132). Erythritol is a four-carbon polyol, which possesses several properties such as sweetness (about 70-80% of sucrose), tooth friendliness, very low calorific value (0.3 kcal/g, a tenth of sucrose), non-carcinogenicity and, unlike other polyols, causes little, if any, gastrointestinal discomfort (Harald and Bruxelles (1993) Starch/Starke 45:400-405).
Traditional industrial erythritol production is carried out by adding catalysts such as hydrogen and nickel to the raw material sugars under the environment of high temperature and high pressure. Another process is performed by the chemo-reduction of raw materials such as meso-tartarate (Kent, P. W., and Wood, K. R. (1964) J. Chem. Soc. 2493-2497) or erythrose (Otey, F. H., and Sloan, J. W. (1961) Ind Eng. Chem. 53:267) to obtain erythritol. In addition, erythritol can be produced by a number of microorganisms. Such organisms include high osmophilic yeasts, e.g., Pichia, Candida, Torulopsis, Trigonopsis, Moniliella, Aureobasidium, and Trichosporon sp. (Onishi, H. (1967) Hakko Kyokaish 25:495-506; Hajny et al. (1964) Appl. Microbiol. 12:240-246; Hattor, K., and Suziki, T. (1974) Agric. Biol. Chem. 38:1203-1208; Ishizuka, H., et al. (1989) J. Ferment. Bioeng. 68:310-314.)
The invention features isolated strains of the Moniliella species with enhanced capacities for the conversion of glucose to erythritol. Such strains can produce erythritol from glucose with a conversion rate of at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or greater under optimal conditions.
Strains of the invention include isolates of Moniliella from a natural source; and the mutants of a Moniliella strains, e.g., a Moniliella strains assigned the American Type Culture Collection (ATCC) accession numbers of PTA-1227, PTA-1228, PTA-1229, PTA-1230, and PTA-1232. One particular mutant strain is the isolated strain, N61188-12, deposited with the American Type Culture Collection with the accession number PTA-2862.
As used herein, the term xe2x80x9cmutantxe2x80x9d refers to a strain whose genetic composition differs by at least one nucleotide, e.g., a substitution, insertion, or deletion, relative to a reference or parent strain. A mutant of the invention can be produced by a number of methods. One method is the selection of strains with increased erythritol conversion rates relative to a parent strain. The strains can be obtained by random mutagenesis of the parent strain, e.g., by means of a chemical mutagen, a transposon, or irradiation. In addition, a mutant strain of the invention can include a recombinant nucleic acid sequence. For example, a mutant may be a strain that harbors an additional nucleic acid sequence, e.g., a sequence transformed, transduced, or otherwise inserted into a cell of the parent strain. The additional nucleic acid sequence can encode a polypeptide that is generally or conditionally expressed. Alternatively, the additional nucleic acid sequence can encode a nucleic acid sequence capable of altering cell physiology, e.g., an anti-sense, a ribozyme, or other nucleic acid sequence. In another instance, the inserted nucleic acid is inserted into an endogenous gene, and alters (e.g., enhances or disrupts) its function. For example, the inserted nucleic acid can be a knockout construct that inactivates the endogenous gene; or an artificial enhancer or promoter that increases transcription of the endogenous gene. The mutation can disrupt the ability of the parental strain to import, assimilate, or consume erythritol or mannitol.
The invention also features a method of producing erythritol. The method includes growing a Moniliella strain of the invention, e.g., an enhanced mutant, in a culture; and purifying erythritol from the culture, e.g., from the supernatant or from the cell pellet.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects and advantages of the invention will be apparent from the description and drawings, and from the claims.
The fungus Moniliella is capable of fermenting simple sugars to produce erythritol, a well-relished component of many cuisines. Screening and mutagenesis are used to identify improved strains of Moniliella that are capable of highly efficient erythritol production yields. Such strains are ideal for large-scale erythritol production, as can be achieved by the exemplary methods described herein.
Isolation of Enhanced Erythritol Producing Strains
Isolates of Moniliella can be obtained from a natural source as described in U.S. patent application Ser. No. 09/585,926, filed Jun. 2, 2000, now U.S. Pat. No. 6,300,107. For example, isolates of Moniliella can be obtained natural sources having high sugar content include honey, preserved fruit, and pollen. Each strain is identified based on its capability to convert glucose to erythritol and its various morphological and physiological traits. As used herein, the xe2x80x9cglucose-to-erythritol conversion ratexe2x80x9d is defined as the amount of erythritol produced divided by the amount of glucose consumed. The resulting ratio can be expressed as a percentage. The glucose-to-erythritol conversion rate of a fungal strain can be calculated by the following method. The strain is first cultured in a 10-ml broth containing 30% glucose and 1% yeast extract (initial cell density 1xc2x7105 cells/ml) in a 50 ml flask in a rotary shaker at 150 rpm and 30xc2x0 C. for 6 days. Then, both the concentration of erythritol in the medium and the concentration of glucose in the medium are determined. The conversion of 1 g of glucose into 0.3 g of erythritol is termed a 30% conversion rate. The morphological traits are determined following growth on 4% malt extract, 0.5% yeast extract agar for 10 days at 20xc2x0 C. See The Yeasts, A Taxonomic Study, Edited by Kurtzman et al., 4th Ed., page 785, Elsevier, Amsterdam (1998)
A mutant of a Moniliella strain can be obtained by the mutagenesis method described in Ishizuka, et al. (1989) J. Ferment. Bioeng. 68:310-314, or a variation thereof (see also U.S. Pat. No. 5,036,011). One variation for the mutagenesis of Moniliella cells with N-methyl-N-nitrosoguanidine (NTG) is described as follows. Moniliella cells are inoculated in broth with 30% glucose and 1% yeast extract, and cultured overnight at 30xc2x0 C. on a rotary shaker at 150 rpm. This culture is diluted 1:100 into 10 ml of broth with 30% glucose, and incubated at 30xc2x0 C. on a rotary shaker at 150 rpm for 1 day. The culture broth is centrifuged at 3,000 rpm for 15 min to form a cell pellet and the supernatant is discarded. The cell pellet is washed with 10 ml of sterile 0.1 M pH 7.0 phosphate buffered saline (PBS). The suspension is centrifuged (3,000 rpm, 15 min) and the supernatant is again discarded. The cells are resuspended in PBS, with 150 xcexcg/ml NTG for 10 minutes.
After treatment with NTG, the Moniliella cells are grown in a glucose solution for 3 hours. The culture is then diluted appropriately and spread onto the medium containing 65% glucose and incubated at 30xc2x0 C. for 6 days. Colonies are selected randomly, inoculated into broth containing 30% glucose, and incubated at 30xc2x0 C. on a rotary shaker at 150 rpm overnight. A 1:100 dilution of the overnight culture is used to inoculate into a 30% glucose solution (10 ml) that is incubated at 30xc2x0 C. on a rotary shaker at 150 rpm for 4 days. The medium from this culture is then centrifuged at 12,000 rpm for 10 min. The supernatant is diluted appropriately and the amount of residual glucose is measured using the DNS method (see below). Cultures with higher glucose consumption (i.e., lower residual glucose) are further analyzed to determine erythritol yield. The HPLC method described below can be used to quantitate erythritol yield. Cultures with indications of elevated erythritol yield are subject to further verification. For example, individual colonies are obtained for the culture, re-grown as described above, and reanalyzed. Selected colonies can be improved by additional rounds of mutagenesis according to these procedures.
Measurement of Residual Glucose
4-day-old culture broth is collected and centrifuged at 12,000 rpm for 10 min. The supernatant is diluted appropriately. 1 ml of each diluted solution is added to 0.5 ml of DNS (dinitrosalicylic acid) reagent. DNS reagents (e.g., a. 1% 3,5-dinitrosalicylic acid (DNS). b. 0.2% phenol; c. 0.05% NaHSO3 or 0.025% Na2S2O3; d. 1% NaOH; e, 0.5% potassium sodium tartrate tetrahydrate) were prepared and used according to method described in Miller, G. L. (1958)Anal. Chem. 31:426-428. The mixture is mixed well and incubated at 100xc2x0 C. for 5 min. After cooling under room temperature, 9 ml water is added and the absorbance at 540 nm (OD540 nm) is determined. The absorbance at 540 nm is used to determine the concentration of glucose by comparison with the standard curve, obtained by measuring pure glucose at various concentrations.
Measurement of Erythritol Concentration
The amount of erythritol in a supernatant can be quantitated by HPLC and TLC, e.g., to determine the erythritol-producing capacity of a strain. HPLC analysis is performed by Hewlett Packard H4033A analyzer on an Ion-300 chromatography column, using 0.1 N sulfuric acid as the flowing phase with a flowing rate of 0.4 ml/min, the temperature being set at 75xc2x0 C. For TLC analysis, the Neissner et al. procedure is followed. (Neissner, et al. 1980. Herstellung, aanalyse und DC-trennung von fettsaure erythritpartialestem. FETTE SEIFEN ANSTRICHMITTEL. 82:10-16.). After rinsing Kieselgel 60F254(Merck) with 4% boric acid, the gel is heated in an incubator at 105xc2x0 C. for 20 minutes before use. The spreading solvent is ethylmethylketone:acetone:water (100: 10:10 by vol.) and the color-developing agent is KMnO4 in concentrated sulfuric acid.
Erythritol purified from a supernatant by HPLC or TLC can be further purified by extraction and then dried under reduced pressure. The further purified product and an erythritol standard are acetylated according to the method of Shindou et al. (Shindou et al. 1989. J. Agric. Food Chem. 37:1474-1476.). Erythritol standards are commercially available, e.g., from Merck, Germany. The resulting sample can be assayed by GC-MS to determine if the re-purified product was identical to that of the standard sample.
Large Scale Production of Erythritol
Following the specific examples provided below, a skilled artisan can optimize erythritol yield of a mutant Moniliella strain by identifying preferred pH, temperature, and carbon source for growth and fermentation. Similar analysis can be used to optimize aeration, stirring speed, culture volume, and culture time.
To produce erythritol on a larger scale, 0.2 ml of Moniliella cells preserved in glycerol are added to 50 ml of broth in a 500 ml flask, and incubated at 30xc2x0 C. on a rotary shaker at 150 rpm for about 24 hours. From this culture, 2 ml are used to inoculate a second 500 ml flask with 50 ml of broth. The second culture is incubated at 30xc2x0 C. on a rotary shaker at 150 rpm for 48 hours. The second culture broth is used to inoculate 2 L of broth in a 5 L fermentor (NBS. Edison, New Jersey, USA). The culture conditions are as follows. Aeration: 1 VVM; stirred speed: 500 rpm; temperature: 30xc2x0 C.; culture period: 5-7 days.
For these purposes, the broth can consist of 30%, 35%, 40%, 45%, or 50% glucose, together and 1% yeast extract. In addition, KM72 and KM72F (Shin Etsu, Shin-Etsu Chemical Co., Ltd. 6-1, Ohtemachi 2-chome, Chiyoda-ku, Tokyo, Japan) can be used as a defoamer.
Purification of Erythritol
Media from the fermentor is centrifuged to separate the culture supernatant from pelleted cells. The supernatant is decolored by passage over active carbon (e.g., powdered carbon as can be obtained from a local supplier). The decolored supernatant is desalted and de-proteinated by consecutive passage of over a cation exchange resin, DIAION, WA30 (Mitsubishi) and an anion exchange resin, AMBERLITE IR120 NA (Rohm and Haas Company). The resulting solution is concentrated with the following apparati: EYELA Rotary Vacuum Evaporator N-N Series; EYELA Waterbath SB-450; and EYELA, Aspirator A-3 (Tokyo Rikakikai Co. LTD). The concentrated solution is crystallized at room temperature. Crystals are optionally washed with or re-crystallized in hydrous alcohol and water (e.g., at 4xc2x0 C.) to remove the trace impurities.
Verification of Erythritol Purification
To confirm the chemical identity of the purified product, the NMR spectra of the purified product is compared to the NMR spectra of a standard, e.g., erythritol purchased from Merck Co. (N.J, USA), or another commercial supplier. The samples are dissolved in 100% D2O and placed in an NMR spectrometer (Bruker AM-500, Germany). The following conditions are used for 1H NMR spectra: 400.135 MHz; pulse length: 4.0 xcexcs; acquisition time: 1.245 sec; pulse delay: 1 sec; chemical shifts: D2O as 0 ppm. The following conditions are used for 13C NMR spectra: 100.536 MHz; pulse length: 5.0 xcexcs; acquisition time: 0.623 sec; pulse delay: 2 sec; chemical shifts: 10 mM DSS as 0 ppm.
A skilled artisan can obtain a fungal mutant of the invention and utilize it to the fullest extent to produce erythritol based on the guidance of the following specific example, which is merely illustrative, and not limitative of the scope of the invention. All publications cited herein are incorporated in their entirety by reference.